Effect of tocopherol and taurine on membrane fluidity of retinal rod outer segments

Exp. E!te lle,~. (1987), 45, 769~776

Effect of Tocopherol and Taurine on Membrane Retinal Rod Outer Segments ,JuLlo

.~[ORAN, P A T R I C I A

~ A L A Z A R AND H g l t b l l N 1 h

Fluidity of

PASANTES-~ORALES*

InSHIU/o de FL~'ioloff{a Celulnr, Uniw, r~idod N a c i o n a l A u t d n o m a de ~lle'xico, A p a r / a d o Posted 7(]-6(]0, 04516 Mdxico D,F,, Mexico (Received 19 December ]!186 a n d accepted 17 J u l y 1987) The deficiet~cyof tattritm aJ~d 0~-tOeol)herol results il~ disturbances of the structure of retinal rod f |ip~)st)mes l)repared with lecithin or with lipids from outer segment membranes was examined by steady-state fluorescence ln)htriz'ttion of diphetu¢l-hexatriene (DPHL a-Tot~ophert)[ increased the DPH anisotropy lmrmn~,ter ii~ tmth preparations. The vitamin modified the breakpoint temperature of Arri~euius plots of I)PH anisotropy, and decreased the activation energy. Tauritle tidied to modify any of the parameters examined in both outer segment membra~es and lecithin liposnmes. Ttmse results sugges~ a stabilizer role tk)r toeophero~ ~n r'~M outer segment membratws. In contrast, the reqttiretnent o f routine to nmintain outei" segt~elx~ struet~re seems unrelated to an effect on tim physical state of membrane lipids. Ke!] word.s: tocophero|: taurine; membrtule fluidity: rod outer segmetlts; fluorescence pt~larizt~tiotx. 1. I n t r o d u c t i o n

P h o t o r e c e p t o r o u t e r s e g m e n t (OS) m e m b r a n e s a r e u n i q u e in t h e i r high p r o p o r t i o n o f p o l y u n s a t u r a t e d f a t t y acids, a c o n d i t i o n w h i c h a p p a r e n t l y p r o v i d e s the fluid m i e r o e n v i r o n n a e n t n e c e s s a r y for c h a n g e s in r h o d o p s i n o c c u r r i n g d u r i n g t h e p h o t o e x c i t a t i o n process (Cone, 1972). T h e liigh level o f u n s a t u r a t i o n m a k e s t h e s e m e m b r a n e s p a r t i c u l a r l y s u s c e p t i b l e to d a m a g e c a u s e d b y lipid p e r o x i d a t i o n , wllicll is k n o w n to occur f r e q u e n t l y u n d e r physiological a n d p a t h o l o g i c a l c o n d i t i o n s in the. r e t i n a (Bazan a n d R e d d y , 1985), resulti~)g in d i s r u p t i o n o f m e m b r a n e s t r u c t u r e . A v a i l a b l e e v i d e n c e sut)ports an involb, e m e n t o f r o u t i n e a n d ce-tocopherol in m e c h a n i s m s relat+ed to t h e m a i n t e n a n c e o f t h e m o r p h o l o g i c a l i n t e g r i t y o f p h o t o r e c e p t o r s . Botl~ c o m p o u n d s are h i g h l y c o n c e n t r a t e d in p h o t o r e c e p t o r s (Orr, C o h e n a n d L o w r y , 1976; F a r n s w o r t t a a n d D w t z , 1976) a n d a d e c r e a s e in t h e i r l.qlysiological levels results in a s e v e r e a l t e r a t i o n o f t h e well-organized s t r u c t u r e o f t h e lamellar discs ( H a y e s , 1974; H a y e s , C a r e y a n d S e h m i d t , 1975; R o b i s o n , K u w a b a r a a n d Bieri, 1979; P a s a n t e s - M o r a l e s , Q u e s a d a , Cdrabez a n d H u x t a b l e , 1983). I n vitro, a d d i t i o n o f t o c o p h e r o l to r e t i n a or isolated OS p r o t e c t s m e m b r a n e s f r o m d a m a g e i n d u c e d b y air e x p o s u r e or hy o x i d a t i v e c o h d i t i o n s ( F a r n s w o r t h m~d D r a t z , 1976 ; S h v e d o v a , S i d o r o v a n d N o v i k o v 1979). T a u r i n e also s h o w s a m a r k e d ability, to p r e s e r v e isolated OS s t r u c t u r e a g a i n s t a n u m b e r o f d e l e t e r i o u s c o n d i t i o n s i n c l u d i n g o x i d a t i o n b y irona s c o r b a t e ( P a s a n t e s - M o r a l e s a n d Cruz, 1984) at~d c o n t i n u o u s i l l u m i n a t i o n ( P a s a n t e s Morales a n d Cruz, 1985). T h e m e c h a n i s m s by w h i c h t o c o p h e r o l a n d t a ~ r i n e p r e s e r v e t h e s t r u c t u r e o f OS are n o t c l e a r so far. T h e p r o t e c t i v e effect o f t o c o p h e r o l m a y r e s u l t f r o m its w e l l - k n o w n a c t i o n as a n a n t i o x i d a n t (Tappel, I980), b u t d i r e c t effects on m e m b r a n e o r g a n i z a t i o n c a n n o t be e x c l u d e d since c h a n g e s on m e m b r a n e s t a b i l i t y a n d p e r m e a b i l i t y i n d u c e d b y * To whom all correspondence should be addressed. 0014-4835/87/120769+08 $03,00/0

@ 1987 Academic Press Limited

770

,1. MORAN, I'.SALAZAR AND H.I'ASANTES-MORAI, ES

t o e o p h e r o l h a v e been o b s e r v e d in b o t h biological a n d artificial m e m b r a n e s (Maggio, Diplock a n d L u c y , 1977; L u c y , 1978; S t e i n e r , 1981 ; M a s s e s , She a n d P o w n a l i , 1982). A possible a n t i o x i d a n t effect o f t a u r i n e as t h e m e c h a n i s m s u b s e r v i ~ g its p r o t e c t i v e a c t i o n on m e m b r a n e s has been ruled o u t b y r e c e n t e v i d e n c e (Pasaz~tes-Morales a n d Cruz, 1984, I985). A l t e r n a t i v e l y , t a u r i n e m i g h t c o n t r i b u t e to preser~'e m e m b r a ~ e s t r u c t u r e by inducing conformational changes within membranes, either by altering t h e physical s t a t e o f t h e m e m b r a n e lipids ( H u x t a b l e a n d Sebring, 1983) or by a c t i n g a t a m o l e c u l a r level o n m e m b r a n e p r o t e i n s ( L o m b a r d i n i , 1985). I n a n y e v e n t , d i r e c t m e m b r a n e i n t e r a c t i o n s o f t a u r i n e a n d locol)herol o f t h e kind s u g g e s t e d a b o v e a r e m o s t p r o b a b l y a f f e c t i n g m e m b r a n e fluidity. T o inve.ntigate t h e s e possibi|ities,in t h e p r e s e n t s t u d y we e x a m i ~ e d t h e effect o f t o e o p h e r o l a n d t a u r i n e on f l u i d i t y o f m e m b r a n e s o b t a i ~ e d f r o m isolated frog rod (ROS) as well as o f artificial vesicles p r e p a r e d with lecithin or |ipids f r o m 0 S m e m b r a n e s . 2. M a t e r i a l s

and Methods

RO8 were isolated fron~ retb~as of dark-adapted frogs, by get, tie vortex'it~g (bll~twed by eentrifl~gatiotl at 1500g. ROS membranes were obtained fl,om ROS lysed by a~ osmotic shock in water. Membrane tipids wel~e extracted aecortlil~g to the procedure of Folch, Less and Sloane-Stanley (19571. Lipid vesicles (|il~osomes } were prepared b y mixing ROS lipids or soybean lecithin (Sigma Chemical C'o.) in a Krebs-Tris buffer eontalni~g It8 m,~l NaCI, 1"2 mM KH2PO4; 4.7 ms1 KCI; 2.5 mm Ca(;12:1-17 m.~ MgSO4:5.6 m.~ gluco.uo at}d 25 m.~t Tris-Ct, pH 7"2. Lipid eoneelltratioll used was 0.4 mg ml -~. Sul)sequent sonicati(m of the lipid suspension yielded a translucent vesicle preparation. Determinations oic steady-state flttorescellt po|arizati(m were made using the probe l.(ldiphex~yl- 1,3,5-lm.x'atriene (I)PI4) a t a final concentration of !/~3~, and ~oadit~g ()f membranes by incubation for 120 rain at 37 °C. Tocopherol, at a com,e,ltration of I J)~mil~ etb~t~o/(0"5 % fiual) or 25 mM taurine was prese~lt during loading in the incubation medium whereas control samples were added with a correspo~dil~g w~lumt- of ethanol or watez,. Preparations of liposotlles or I~ler~bralles withotlt DPH g~ve a ~,,7,!igible ha(.kgrouud fl~orescen(.e (less than 5%). Polarizatia~ measurements we~'e made as a functi(m of t e m p e r a t u r e i~ a l)olariz~tion spectrofluorometer (SLM Instruments, model SMC 210) using an excitation wavelength of :{65 nm selected by a monochromator, at~([ a~ emission wavelength of 428 nm selected by filters. Cuvette t e m p e r a t u r e was maintained by a circulating water-t)ath.-Tl~e T-ft)rmat method was used, so t h a t t)aral le] and l)erl')end i euh~~' (,oml)one~) ts ~tr() obser~'(,(l sitn (dt atmously through separate channels. 3. : R e s u l t s

R e s u l t s are e x p r e s s e d as t h e D P H a n i s o t r o p y p a r a m e t e r , [(ro/r--1)1 -~. D P H is a f l u o r o p h o r e which i n c o r p o r a t e s in t h e h y d r o c a r b o n region o f t h e m e m b r a n e , s e r v i n g as ~t c o n v e n i e n t p r o b e o f t h e f l u i d i t y o f t h e lipid e n v i r o n m e n t i~ w h i c h it resides (Shinitzki a n d B a r e n h o l t z , 1974). D P H [ ( r J r ) - - I I -~ is i n v e r s e l y p r o p o r t i o n a l t~ t h e a p p a r e n t r o t a t i o n a l r e l a x a t i o n t i m e o f t h e p r o b e a n d p r o v i d e s an i n d e x ~¢~the fluidity o f lipid molecules in t h e m e m b r a n e b i l a y e r ( B r a s i t u s a n d S c h a c h t e r . (980). T h e v a l u e of t h e m a x i m a l l i m i t i n g a n i s o t r o p y (r0) o f I ) P H was t a k e n as 0"362 ( S h i t d t z k i eu~d B a r e n h o l t z , 1974). T h e a c t i v a t i o n e n e r g y for a n i s o t r o p y p a r a m e t e r (El was calculated f r o m t h e slope o f t h e A r r h e n i u s plot, fit by t h e m e t h o d o f least s q u a r e s usit~g a computer program. T h e d e g r e e o f fluorescence p o l a r i z a t i o n o f I ) P H was c o n s t a n t a f t e r i n c u b a t i o n of R O S w i t h D P H for 30 rain a t 37 °C, a n d o v e r a r a n g e o f 1 - 1 0 / t ~ . T h e c o n c e n t r a t i o n o f D P H - l a b e l e d m e m b r a n e s or vesicles was a l w a y s in a r a n g e w h i c h e x c l u d e s depolarization due to light scattering.

! 9"1 +_0"~ 15'9_+,0'65~

Liposomes (:ontrol Toeopherol 1-91_.+0.02 1,72_40,02~

2"564-0.08 _ . 1"i0+0.12§ 2.72 ± 0' 10 2.40~ 0.05"}"

t.44 ±0'07 r, , {n,s.) 1,3,)±0"03

Breakpoint

1'63 _+0'02 2-29+_0"tS:~

1"194-0"07 1"23_+0"02{n.s.)

10 °C

0"95± 0.02 1.30 ~0,19"[

0"64+ 0.04 0"84_+0"02~

37 °(!

Anistropy parameter

* Activation energy above and below the breakpoint is expressed in keal tool~. Membranes were obtained from isolated ROS as described in Materials and Methods. I~iposomeswere prepared ~ritb lipids extracted from ROS membranes. The concentration of toeopherol was 1 m.~t. Breakpoint and activation energy values were calculated from slopes fit by least squares using a computer program. Values are means_.+s.n, of 4-fi experiments, t P < 0'02; ~:P < 0"01; §P < 0"(t01, compare(/with each control value.

21'9 + 0'48 IJ(, 1_+.+0.-.) ~-~"

~lemt~ranes Control 'l'ocopherol

°C

l)reparatkm l~reakpoint Activation energy* Above Below

Effect of tocopherol on temperature depe~zdence of DPH fl~wre~cence ani~otropy of o~tterseffment membranes and liposomes

TAnL~: I

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J. MORAN,

P. S A L A Z A R

AND

H. P A S A N T E S - M O R A I ~ E S

T(°C) 35 O'10

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I"1(;. I. A r r h e n i u s p l o t of I ) P H a n i s o t r o p y p a r a m e t e r in c o n t r o l ( Q ) a n d a~-toeopherol-loaded (C)) R O S m e m b r a n e s . E a c h p o i n t r e p r e s e n t s t h e m e a n o f five s e p a r a t e e x p e r i m e n t s . S t a n d a r d e r r o r s were a l w a y s less t h a n I0 d~j. C u r v e s were fit te(l b y l e a s t - s q u a r e a n d t h e coefficiellt c o r r e l a t i o n v a l u e s were in tile range 0"9978-0"9998.

T{°C 45

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Fle,. 2. A r r h e n i u s p l o t o f D P H anisotrol~v p a r a m e t e r in e o n t r o l ( O ) a n d a - t o e o p h e r o l - l o a d e d ( O ) l i p o s o m e s p r e p a r e d w i t h lipids from I l O S m e m b r a n e s . E a c h p o i n t r e p r e s e h t s t h e m e a n o f f o u r s e p a r a t e e x p e r i m e n t s . S t a n d a r d e r r o r s were a l w a y s less t h a n I 0 ° ~ . Curves were f i t t e d b y l e a s t - s q u a r e a n d t h e coefficient c o r r e l a t i o n v a l u e s were in t h e r a n g e 0-9979-0"9981.

TO(3Ol'HlgltOI.,, TAURINE

AND RETINAL

773

('C) 15

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35

OS F L U I D I T Y

25

0.10

0.05

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/-

~-~-o.o5 0

~

-0.10

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/

3.2

3,3

3.4 lIT

3.5

( ' K - ' x l O "~)

Flo. 3. Arrhenius plot of I ) P H anisotropy parameter in control (Q) and taurine-exposed (O) ROS membranes. Each point represents the mem~ of four separated experiments. Standard errors were ahvays less than 10%. T ("C) 3,5 't

2,5 I

--I

15 .....

J

............ ; '

!

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L.~ -0.48 O

-0.,58

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t

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3.4

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-

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(°K-" x l O ~)

FIG. 4. Arrhenius plot of D P H anisotrol)y parameter in control ( 0 ) and taurine-exposed (O) lecithin liposomes. Each I~oint represents the mean of four separated experiments. Standard errors were always less than 10%.

774

J.M()RAN, |'.SALAZAR AND H, I'ASANTES-MORAI, ES

The variation of D P H [(r0/r)-- 11-! with teml)erature in the range of 5-4(i °C was d e t e r m i n e d for ROS membranes and for lil)osomes of lecithin or ROS m e m b r a n e lipids. At all t e m p e r a t u r e s examined, the I)PH anisotropy value was slightly lower for ROS m e m b r a n e s than for" ROS ]il)id vesicles. For instance, at 1(1 °C the anisotropy value was 1"!9 and 1"63 for ROS membranes and ROS vesicles, respectively. At 37 °C, these values were 0"64 and 0"95 (Table l). These results suggest t h a t the rotational fi'cedom of the l)robe is not restricte(l in the intact ROS memlwane. Arrhenius plots of the wtriation of I)PH [(r0/r)-- I1-1 in ROS membranes and R()S m e m b r a n e lipid vesicles arc shown in Figs I and 2. Data for both preparations fit best a two-slope plot with breakt)oints of 19"I °C for lil)osomes f~.'om lipid membranes and 21"9 °C for the whole mtmbr'ane.~ (q able ). q kc ? d d tion of' ethanol necessary fbr solubilizing tocopherol did not sighificantl5 modif:~ the Arrhenius plot nor an5 of the lmrametevs examined (compare control curves of Figs I and 3). T r e a t m e n t with tocopherol increased the 1)PH anisotropy parameter at all tested temperatures in the two preparations (Figs I, 2). In addition, tocopherol altered the breakpoint t e m p e r a t u r e from 19"1- to 16"(1°C in liposomes fi'om lipid membranes, an(i from 21"9- to 19"1 °C in the whote-menlbranc preparation (Table 1). The slope of Arrhenius l)lots is a measure of E, the fusion (or flow) activation energy. This is an expression which characterizes the degree of order in lipid-lil)id intevaetions; a lower value of E in(ticates a h!gh degree of order. A(:tivatlon energy values in the presence of" to(.ol)herol were reduced below and above the breaklmint t)3' 6- and 57[¼~ for m e m b r a n e s and 12- and 10% for liposomes (Table I). Taurine, at a concentration of 25 m~t, failed to too(lily any of the parameters examined, i.e. breakpoint temperatures or the slope of the Avrhenius curves. This was observe(t both in ROS membranes (Fig. 3) and in lecithin liposomes (Fig. 4). 4. D i s c u s s i o n

The results of the t)resent s t u d y show that tocopherol is able to modit~y the physical properties of I{OS membrane, increasing the rigidity as well as the degree of order of m e m b r a n e lipids. These effects of tOCol)herol show-some similarities with those of cholesterol, one of the best known examples of a m e m b r a n e stabilizer', which also increases the degree of order of membranes (Ladbrooke, Williams and Chapman, 1968; Shiga and Maeda, 1980). Some of the biological actions of tocophevol m a y be mediated by its fun(.tion as a n t i o x i d a n t (Tappel, 1980), but there is als0 evidence t h a t tocopherol may act directly through an intsraction with m e m b r a n e s (Maggio, l)iplock and Lucy, 1977; Lucy, 1978; Steiner, 1981; Massey, She and Pownall, 1982). Tocopherol inhibits platelet aggregation, a process closely related to the physical state of the membrane. The observation t h a t the same effect is produced by the oxidized metai)olite tocopheryl quinone is against the interpretation t h a t this effect involves an a n t i o x i d a n t action. Also, in cultured human lymphoblastoids, tocopherol shows a protective effect against m e m b r a n e d a m a g e p r o d u c e d . b y retinol, under conditions in which oxidation is not occurring (Pasantes-Morales, Wright and Gaull, 1984). A number of reports in liposomes also point out on a m o d u l a t o r y effect of tocopherol on the structure and permeability of the lipid bilayer (Maggio, l)iplock and Lucy, 1977; Diplock, Lucy, Verrinder and Zieleniewski, 1977; Lucy, 1978; Massey, She and Pownall, 1982; Stilwell and BILvant, 1983) This kind of m e m b r a n e interaction of tocopherol may account for some of the consequences of its deficiency observed in vivo, such as e r y t h r o c y t e hemolysis

T O C ( ) I ' H E R O L , TAUI{~INE AND R E T I N A L OS F L U I D I T Y

775

( L e o n a r d a n d L o s o w s k y , 1971) a n d p h o t o r e c e p t o r d i s r u p t i o n ( R o b i s o n , K u w a b a r a a n d Bieri, 1979). W i t h o u t t h e v i t a m i n , m e m b r a n e s m a y h a v e a n a b n o r m a l l y h i g h p e r m e a b i l i t y a n d be s u b j e c t o f d e g r a d a t i v e p r o c e s s e s as well as b e i n g p a r t i c u l a r l y s u s c e p t i b l e to d a m a g e . T a u r i n e s h o w s a n u m b e r o f effects on cell m e m b r a n e s , p a r t i c u l a r l y a t e x c i t a b l e tissues. Besides its w e l l - k n o w n p r o t e c t i v e effects on p h o t o r e c e p t o r m e m b r a n e s , t a u r i n e c o u n t e r a c t s m e m b r a n e d a m a g e c a u s e d by e x t e r n a l a g e n t s to t h e h e a r t ( K r a m e r , C h o v a n a n d Shaffer, 1981) to h e p a t i c cells ( N a k a s h i m a , T a k i n o a n d K u r i y a m a , 1983), a n d to h u m a n ] y m p h o b l a s t o i d s ( P a s a n t e s - M o r a l e s , W r i g h t a n d Gaul], 1984, 1985). F r o m t h e r e s u l t s o f t h e p r e s e n t s t u d y it c l e a r l y e m e r g e s t h a t t a u r i n e effects are n o t m e d i a t e d t h r o u g h c h a n g e s in m e m b r a n e p r o p e r t i e s r e s u l t i n g in a m o d i f i c a t i o n o f m e m b r a n e fluidity. A l t e r n a t e possibilities, i n c l u d i n g a n effect on ionic fluxes or on p r o t e i n c o n s t i t u e n t s in t h e m e m b r a n e s h o u l d be c o n s i d e r e d . ACKNOWLEI)GM

ENTS

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